Hepatitis C virus (HCV) infection afflicts approximately 170 million people worldwide and chronic virus replication in these individuals is a significant cause of liver inflammation, cirrhosis, hepatocellular carcinoma, and a host of extra-hepatic disease states. No vaccine exists to prevent transmission of this blood borne pathogen, and the current non-specific drug therapy used to treat patients is of limited efficacy for the mos common HCV genotypes. Although progress has been made in the last decade in the development of specific small molecule inhibitors targeting a number of viral proteins, the error prone nature of HCV RNA replication suggests resistance to these inhibitors will develop. A cocktail-based therapeutic approach using multiple specific inhibitors targeting distinct aspects of the HCV life cycle is therefore a prudent strategy for future therapeutics, and this approach requires the development of numerous specific inhibitors with unique mechanisms of action. Despite an essential role in the virus life cycle, the viral NS2 protease has seen surprisingly litle activity as an anti-viral target. NS2 is a papain-like cysteine protease that catalyzes the auto-catalytic cleavage of the NS2/NS3 junction in the viral polyprotein, and this cleavage is essential for productive infections. Using a series of engineered autonomously replicating HCV replicons, we have developed a potential screen to identify compounds that disrupt viral replication in a manner dependent on NS2 activity. Although preliminary results suggest the large format version of this assay can identify NS2 inhibitors, considerable effort will be required before this assay can be moved into a high content screening environment. This application is directed towards announcement PA-10-213, a program specifically geared towards assay development for eventual access to the NIH MLPCN screening center program. The overall goal of this proposed project is to develop our assay for NS2 function into an assay appropriate for high throughput small molecule screening and develop appropriate counter and secondary screens to maximize the impact of future screening efforts. To this end we have proposed a series of logical experiments in three specific aims to miniaturize and validate our assay, develop appropriate counter screens that fit into a logical compound triaging plan, and provide a context in which tool compounds that result from our screen might be used to understand HCV biology.